The goal of this proposal is to understand how messenger RNA degradation is initiated in the Gram-positive bacterium Bacillus subtilis. Rapid turnover of bacterial mRNA is an important element in regulating gene expression and allows quick adaption of microorganisms to changing environmental conditions. From recent reports concerning mRNA decay in Escherichia coli, as well as our own work with B. subtilis, a general model for the initiation of mRNA decay can be proposed. Briefly, the rate-determining step in decay of most mRNA molecules is the binding of an endonuclease at or near the 5' end of the message. The endonuclease then migrates downstream and cleaves at an internal site to initiate overall decay. Past research in this laboratory has concentrated on the decay of mRNA encoded by the erythromycin resistance gene ermC. Stabilization of ermC mRNA is achieved by ribosome stalling near the mRNA 5' end, which prevents ribonuclease binding. Our work on ermC mRNA decay has led us to propose the following ways to study initiation of mRNA decay in B. subtilis: 1. The nuclease that binds to the 5' end of ermC mRNA and initiates decay will be characterized through the use of substrates that are designed to detect a 5'-binding nuclease activity. The 5'-binding nuclease will be purified from B. subtilis extracts, an N-terminal amino acid sequence will be determined, and oligonucleotide probes will be used to identify a clone containing the gene encoding this ribonuclease. 2. We have shown that mRNA decay can initiate from an internal site that is cleaved by "Bs-RNase III", a ribonuclease that resembles E. coli RNase III. We plan to isolate this nuclease and clone the gene that encodes it in order to assess its role in RNA processing. 3. The stabilizing effect of a hairpin structure at the 5' end of a B. subtilis message will be determined. The presence of an RNase E-like activity in B. subtilis will be tested by two different methods. A genetic technique to select for DNA fragments that encode endonuclease cleavage sites is proposed.